Masahito Asada

Distribution and ecological aspects of sand fly (Diptera: Psychodidae) species in Sri Lanka.

ABSTRACT: n Human indigenous cutaneous leishmaniasis caused by Leishmania donovani complex is endemic in Sri Lanka. We performed an entomological survey to determine the distribution of probable vector species. Sand flies were collected in districts in the dry zone, in the wet zone highlands, and in the wet zone coastal belt of Sri Lanka using CDC light traps, sticky traps and cattle-baited net traps during July, 2005. The survey was reconducted in February, 2006. Overall, 584 sand flies belonging to Phlebotomus (266 specimens, 2 species) and Sergentomyia (318 specimens, 8 species) genera were collected. A total of 266 Phlebotomus was identified as P. argentipes (258/266; 97%) and P. stantoni (8/266; 3%). The identification studies of Sergentomyia specimens showed that there are at least 8 species in Sri Lanka. Higher number of Phlebotomus sand flies (76/266) were caught in the southern part of the country compared to the other parts probably due to different ecological aspects. P. argentipes were widely distributed throughout the island whereas P. stantoni were collected only in four districts. Since P. argentipes is known to be the vector of L. donovani responsible of visceral leishmaniasis in India, this species may be incriminated as the most possible vector of human cutaneous leishmaniasis in Sri Lanka.

Ungulate malaria parasites

Haemosporida parasites of even-toed ungulates are diverse and globally distributed, but since their discovery in 1913 their characterization has relied exclusively on microscopy-based descriptions. In order to bring molecular approaches to bear on the identity and evolutionary relationships of ungulate malaria parasites, we conducted Plasmodium cytb-specific nested PCR surveys using blood from water buffalo in Vietnam and Thailand, and goats in Zambia. We found that Plasmodium is readily detectable from water buffalo in these countries, indicating that buffalo Plasmodium is distributed in a wider region than India, which is the only area in which buffalo Plasmodium has been reported. Two types (I and II) of Plasmodium sequences were identified from water buffalo and a third type (III) was isolated from goat. Morphology of the parasite was confirmed in Giemsa-reagent stained blood smears for the Type I sample. Complete mitochondrial DNA sequences were isolated and used to infer a phylogeny in which ungulate malaria parasites form a monophyletic clade within the Haemosporida, and branch prior to the clade containing bird, lizard and other mammalian Plasmodium. Thus it is likely that host switching of Plasmodium from birds to mammals occurred multiple times, with a switch to ungulates independently from other mammalian Plasmodium.

Adhesion of MRP8/14 to amastigotes in skin lesions of Leishmania major-infected mice

In murine experimental cutaneous leishmaniasis, parasite infection induces an accumulation of macrophages expressing migration inhibitory factor-related protein 8 (MRP8) and MRP14, two members of the S100 calcium-binding protein family. Although MRP8 and MRP14 are cytoplasmic proteins expressed by myeloid cells, recent studies have demonstrated that MRP8 and MRP14 have extracellular functions such as chemotactic activities. In this study, we examined whether extracellular MRP8 and MRP14 interact with Leishmania parasites during infection. By immunohistochemistry, positive staining by MRP8 and MRP14 was detected on amastigotes in skin lesions of Leishmania major-infected mice. Western blot analysis with amastigotes purified from the skin lesions demonstrated that both of these proteins adhered to amastigotes. The adhesion of MRP14 to amastigotes was reproduced in vitro and enhanced in the presence of Ca2+ and Zn2+. MRP14 adhered to not only amastigotes, but also promastigotes, suggesting receptor molecules for MRP14 are expressed commonly in both developmental stages.

Studies of trypanosomiasis in the Luangwa valley, north-eastern Zambia

BackgroundThe present study, conducted in Zambia’s Luangwa valley where both animal African trypanosomiasis (AAT) and human African trypanosomiasis (HAT) are endemic, combined the use of microscopy and molecular techniques to determine the presence of trypanosome species in cattle, goats and tsetse flies.MethodsThis study was conducted between 2008 and 2010 in Petauke, Chama and Isoka districts, north-eastern Zambia. A total of 243 cattle, 36 goats and 546 tsetse flies, were examined for presence of trypanosome species using microscopy, PCR and loop-mediated isothermal amplification (LAMP).ResultsThere was poor agreement among the test methods used for detection of trypanosomes species in animal blood and tsetse flies. Trypanosomes were observed in 6.1xa0% (95xa0% CI: 3.3-8.9xa0%) of the animals sampled by microscopy, 7.5xa0% (95xa0% CI: 4.4–10.6xa0%) by PCR and 18.6xa0% (95xa0% CI: 13.6–23.6xa0%) by PFR-LAMP. PFR-LAMP was more sensitive for detecting Trypanozoon than KIN-PCR. The highest occurrence of AAT was recorded in cattle from Petauke (58.7xa0%, 95xa0% CI: 44.7–72.7xa0%) while the lowest was from Isoka (5.4xa0%, 95xa0% CI: 0.8–10.0xa0%). Infection of both cattle and goats with Trypanosoma congolense and T. vivax was associated with clinical AAT.ConclusionWhen selecting molecular techniques for AAT surveillance in endemic regions, the KIN-PCR and species-specific PCR may be recommended for screening animal or tsetse fly samples for T. congolense and T. vivax, respectively. On the other hand, species-specific PCR and/or LAMP might be of greater value in the screening of animal and human body fluids as well as tsetse fly samples for Trypanozoon.

Adenosine–uridine-rich element is one of the required cis-elements for epimastigote form stage-specific gene expression of the congolense epimastigote specific protein

It is known that gene expression in kinetoplastida is regulated post-transcriptionally. Several previous studies have shown that stage-specific gene expression in trypanosomes is regulated by cis-elements located in the 3 untranslated region (UTR) of each mRNA and also by RNA binding proteins. Our previous study revealed that gene expression of congolense epimastigote specific protein (cesp) was regulated by cis-elements located in the 3UTR. In the present study, we identified the adenosine and uridine rich region in the cesp 3UTR. Using transgenic trypanosome cell lines with different egfp expression cassettes, we showed that this adenosine and uridine rich region is one of the regulatory elements for epimastigote form (EMF) stage-specific gene expression via the regulatory cis-element of the eukaryotic AU rich element (ARE). Therefore this required element within the cesp 3UTR was designated as T. congolense ARE. This required cis-element might selectively stabilize mRNA in the EMF stage and destabilize mRNA in other stages. By RNA electro mobility shift assay, unknown stage-specific RNA binding proteins (RBPs) whose sequences specifically interacted with the required cis-element were found. These results indicate that EMF stage specific cis-element and RBP complexes might specifically stabilize cesp mRNA in EMF.

The epimastigote stage-specific gene expression of CESP is tightly regulated by its 3' UTR.

It is known that gene expression in kinetoplastids is regulated post-transcriptionally. Although previous studies have shown that stage-specific gene expression in trypanosomes is regulated by cis-elements located in the 3 untranslated region (UTR) of mRNA and also by RNA binding proteins that use bloodstream and procyclic forms, no studies have been performed in the epimastigote form (EMF) of African trypanosomes. This study shows that the cis-elements of the congolense epimastigote-specific protein (cesp) gene regulate its EMF-specific expression. Four different egfp expression cassettes containing 5 and 3 UTRs derived from actin and cesp were integrated into the Trypanosoma congolense genome. EGFP expression was observed in EMF trypanosomes when the egfp cassette contained the cesp 3 UTR. These results indicate that the essential cis-element located in the cesp 3 UTR selectively stabilizes mRNA in the EMF stage and/or destabilized mRNA in other stages. This is the first report of post-transcriptional gene expression regulation in EMF African trypanosomes.

Serological characterizations of tandem repeat proteins for detection of African trypanosome infection in cattle.

Serological diagnosis is a useful method to detect African trypanosome infection in livestock animals. Currently available serological tests utilize whole parasites or crude antigens, and recombinant antigens may improve reproducibility/standardization and reduce production costs. With a goal of identifying such recombinant proteins, we computationally identified proteins with tandem repeat (TR) domain from the parasite proteomes and evaluated their potential for serological diagnosis of African trypanosome infections in cattle. Among those tested, Tbg4 demonstrated the best performance with 92% sensitivity, followed by TbbGM6 (85%), TcoGM6 (85%), Tbg2 (65%) and Tbg5 (65%). Although further evaluations such as investigating cross-reactivity to other infections are needed, our data indicate the potential of these antigens for detection of African trypanosome infection in cattle.

A PCR-based survey of animal African trypanosomosis and selected piroplasm parasites of cattle and goats in Zambia.

We screened cattle and goats from the districts of Chama, Monze and Mumbwa in Zambia for animal African trypanosomes, Babesia bigemina and Theileria parva using PCRs; 38.1% of the samples tested positive for at least one of the parasite species. The most common parasite was Trypanosoma vivax (19.8%). Its incidence was significantly higher in goats than in cattle, (P<0.05). B. bigemina was found in samples from all the three areas, making it the most widespread of the parasites in Zambia. Among the tested samples, 12.0% of the positive samples were mixed infections. There were significant differences in the infection rates of T. vivax (Mumbwa had a significantly higher infection rate [39.6%, P<0.0001]), Th. parva (Monze had the only cases [P<0.0004]) and B. bigemina (Monze had a significantly higher infection rate [40.5%, P<0.0001]). According to the hematocrit values, the packed cell volume (%) among the cattle with mixed infections was significantly lower than that of the other cattle. The presence of multiple parasite species and mixed infections among the Zambian cattle and goat populations is of both clinical and economic importance to livestock farming. The absence of trypanosomosis among the samples from Monze can be attributed to tsetse eradication efforts that took place around Lake Kariba. This shows that the prevention and control of these parasitic diseases can have a significant impact on the disease status, which can translate directly into the improvement of the livestock sector in Zambia.

Genetic homogeneity of goat malaria parasites in Asia and Africa suggests their expansion with domestic goat host

Plasmodium was first identified in a goat in Angola in 1923, and only recently characterized by DNA isolation from a goat blood sample in Zambia. Goats were first domesticated in the Fertile Crescent approximately 10,000 years ago, and are now globally distributed. It is not known if the Plasmodium identified in African goats originated from parasites circulating in the local ungulates, or if it co-evolved in the goat before its domestication. To address this question, we performed PCR-based surveillance using a total of 1,299 goat blood samples collected from Sudan and Kenya in Africa, Iran in west Asia, and Myanmar and Thailand in southeast Asia. Plasmodium DNA was detected from all locations, suggesting that the parasite is not limited to Africa, but widely distributed. Whole mitochondrial DNA sequences revealed that there was only one nucleotide substitution between Zambian/Kenyan samples and others, supporting the existence of a goat-specific Plasmodium species, presumably Plasmodium caprae, rather than infection of goats by local ungulate malaria parasites. We also present the first photographic images of P. caprae, from one Kenyan goat sample.

Polymorphism of Leishmaniasis Caused by Leishmania donovani Sensu Lato in Asia

Visceral leishmaniasis (VL), also called kala-azar (KA), is the most severe form of leishmaniasis and often fatal if not treated. The causative species of VL are members of Leishmania donovani sensu lato (s.l.), which includes L. donovani, L. infantum, L. archibaldi, and L. chagasi. Its principal victims are children and young adults. Control of KA is urgently needed worldwide. However, due to the diversity of the epidemiological features in each endemic focus, it has not been possible to devise a universal control strategy applicable to all foci. In order to construct a framework of epidemiological, pathological, and taxonomic understanding, and explain how Leishmania species develop different clinical and epidemiological features, further studies on the pathogenesis of different forms of KA and comparative studies of the parasites from different foci are clearly necessary. This chapter summarizes the recent advances in understanding the polymorphism of leishmaniasis as caused by Leishmania donovani s.l. in Asia.